A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments

Abstract

Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly β-citraurin (3-hydroxy-β-apo-8'-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and Mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of β-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in β-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and β-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7',8' double bond in zeaxanthin and β-cryptoxanthin, confirming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7',8' double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration.

Keywords: Apocarotenoid; Citrus; carotenoid; carotenoid cleavage dioxygenase; fruit coloration.; β-citraurin.

Fig. 1.

Structure of the main C₃₀ apocarotenoids identified in citrus fruits (A, β-citraurin; B, β-apo-8′-carotenal) and the potential in vivo precursors (C, zeaxanthin; D, β-carotene; E, β-cryptoxanthin). The asymmetric cleavage site at the 7′,8′ double bonds is indicated. In (F), the different coloration of three different mandarin fruits with a similar C₄₀ carotenoid composition in the peel but displaying marked difference in the C₃₀ apocarotenoid β-citraurin content is shown: null content, a clementine mutant (39E7, Rios et al., 2010); medium content, a clementine (Gross, 1987; Rios et al., 2010); high content, a mandarin hybrid (Fortune; Saunt, 2000).

Fig. 2.

Alignment of Citrus clementina CCD4-like proteins (A) and phylogenetic tree of Citrus CCD4-like and other plant CCDs (B). (A) The alignment of Citrus CCD4-like proteins was created using the CLUSTAL W program (Thompson et al., 1994). Numbers on the right denote the number of amino acid residue. Residues identical for all the sequences in a given position are in white text on a black background, and 75–100% homologous residues are presented on a grey background. The asterisks indicate the histidine residues involved in the coordination of the catalytic Fe²⁺ and black dots indicate aspartate or glutamate residues which are predicted to be fixing the iron atom. (B) The phylogenetic tree was generated based on the alignment of deduced amino acid sequences of C. clementina CCD4-like proteins and other plant CCDs. The tree was constructed on the basis of the Neighbor–Joining method (Saitou and Nei, 1987). The bootstrap values on the nodes indicate the number of times that each group occurred with 1000 replicates. The sequences used to generate the phylogenetic tree and their accession numbers are as follows: Citrus sinensis CsCCD1 (accession no. BAE92958); Arabidopsis thaliana AtCCD1 (accession no. AT3G63520), AtCCD4 (accession no. O49675), AtCCD7 (accession no. NP_195007), and AtCCD8 (accession no. NM_130064); Crocus sativus CCD1 (accession no. CAC_79592), CCD4a (accession no. EU523662), and CCD4b (accession no. EU523663); Citrus clementina CcCCD4a (accession no. Ciclev10031003m), CcCCD4b1 (accession no. Ciclev10028113m), CcCCD4b2 (accession no. Ciclev10030384m), CcCCD4c (accession no. Ciclev1001335m), and CcCCD4d (accession no. Ciclev10013726m); Chrysanthemum morifolium CmCCD4a (accession no.AB247148) and CmCCD4b (accession no. AB247160); Osmanthus fragans OfCCD4 (accession no. EU334434); and Rosa damascena RdCCD4 (accession no. EU334433).

Fig. 3.

Expression of CCD4a, CCD4b1, and CCD4c genes in different vegetative and reproductive tissues of Navel sweet orange (Citrus sinensis). For each gene, the expression values are relative to the sample with the maximum expression level which was set to 1. The data are means ±SD of three experimental replicates.

Fig. 4.

Transcript levels of the CCD4b1 gene and the accumulation of the C₃₀ apocarotenoid β-citraurin, in the peel and pulp of Navel sweet orange (A), Clementine mandarin, and the hybrid Fortune mandarin (B) during fruit development and ripening. Changes in the concentration of the putative precursors of β-citraurin, β-cryptoxanthin, and zeaxanthin, are also shown, and for comparative purposes the same scale for both xanthophylls is used. The stages of fruit development and ripening are: IG, immature green; M1 and M2, mature green; B1 and B2, breaker; C, coloured; and FC, fully coloured fruit, as described in Alquézar et al. (2008b ). Expression values are relative to transcript levels obtained in the peel of sweet orange at the IG stage which was arbitrarily set to 1. Data of carotenoid content and transcripts accumulation are means ±SD of three replicates.

Fig. 5.

Effect of a continuous ethylene treatment (A) and heat treatment (37 ºC) followed by ethylene application (B) on the expression of the CCD4b1 gene in peel of Navel oranges or Clementine mandarin. (A) Mature-green fruits (harvested in October) of Navel sweet orange and Clementine mandarin were exposed to air or to ethylene (10 µl l^–1) at 20 ºC for up to 3 d. (B) Coloured Navel sweet oranges (harvested in November) were incubated at 20 ºC (control) or heat treated at 37 ºC and 90% relative humidity for 3 d. After heating at 37 ºC, fruits were exposed to an ethylene treatment (10 µl l^–1) at 20 ºC for an additional 4 d. The images are representative of the external colour of the fruits at the beginning and the end of each treatment. Expression values are relative to transcript levels obtained in the peel of Navel sweet orange fruits at the onset of the experiments, which was arbitrarily set to 1. N.d. indicates not detected. Data of transcripts levels are means ±SD of three replicates. (This figure is available in colour at JXB online.)

Fig. 6.

Best 3D model of CCD4b1 using the VP14 (PBD: 2biwA) structure from maize as template. The α-helix (α1, α2, and α3) and β-propeller domains are shown in model view (A). A top view of the model allows the visualization of the propeller blades and the Fe²⁺ in the reaction centre (B). White arrows in (B) indicate the location of the four histidines coordinating the Fe²⁺. (This figure is available in colour at JXB online.)

Fig. 7.

HPLC analysis of the in vitro enzymatic activity of Citrus CCD4b1. Assays were incubated for 1h, except for lycopene which was incubated for 6h. The crude lysate of thioredoxin-CCD4b1-expressing E.coli cells (CCD4b1) converted (A) β-carotene into β-apo-8′-carotenal (P1), (B) β-cryptoxanthin into P1 and β-citraurin (P2), (C) zeaxanthin into P2, (D) lutein into 3-OH-ε-apo-8′-carotenal (α-citraurin) (P3), and (E) lycopene into apo-8′-lycopenal (P4) and apo-10′-lycopenal (P5). UV-Vis spectra of obtained products are depicted in the insets. No conversion was observed with the corresponding controls corresponding to crude lysates of thioredoxin-overexpressing cells (Con).